The invention relates to the object characterized in the claims, i.e., microparticles produced from biopolymers, monomers capable of polymerization, active ingredients and/or diagnostically detectable components, a process for their production as well as their use in diagnosis and treatment, especially as contrast media in ultrasonic diagnosis.
It is known that particles, whose diameter is smaller or in the range of the size of red blood cells, can circulate within the circulatory system after injection in the circulatory pathway. Pharmaceutical preparations of such microparticles are thus suitable as vehicle systems injectable in the circulatory system for active ingredients or diagnostic agents in medicine. As vehicle materials, in principle all biodegradable, compatible and non-water-soluble substances are suitable. So far, above all fats, waxes, lipids (e.g., soybean lecithin), denatured biopolymers (e.g., albumin, gelatin) and synthetically biodegradable polymers (e.g., polylactic acid, polyhydroxybutyric acid, polyalkylcyanoacrylates, poly-L-lysine) are described.
There is a difference in how quickly and in the numbers in which the microparticles circulating in the circulatory pathway are recognized, as a function of their physical and chemical properties, by the cells of the monocytic phagocytizing system (MPS) and taken up (mainly in the liver, lung and spleen). The particle charge, the particle size, the properties (molecular weight, amphiphilia) on the particle surface of adsorbed substances, as well as the affinity of the particle surface for blood components such as fibronectin, albumin, etc. are considered as essential factors, which determine the kinetics of the absorption of the microparticles by the cells of the MPS. By specific variation of the physicochemical surface properties of microparticles, the kinetics of the phagocytosis can be influenced by the cells of the MPS and the extent of the concentration of the particles within the corresponding organs (i.a., liver, lung, spleen, bone marrow) (passive targeting). A specific concentration of microparticles in target tissues or body structures, which are not among the organs of the RES, is in this way not possible. It can rather be achieved only by the combination of the microparticles with substances which have site-specific, structure-specific or tissue-specific binding properties (homing devices). But the particles previously described for use in ultrasonic diagnosis are suitable only insufficiently as preparations suitable for the combination with homing devices.
Thus, it has to be accepted in the case of the contrast media described in EP 0 458 079 and DE 38 03 972 that they can be produced only with the help of expensive processes, which make necessary the use of organic solvents, whose use is harmful for reasons of protection of the environment and work place safety. In addition, before using the preparations, there has to be assurance that the used organic solvents are no longer contained in the product to be used pharmaceutically. Moreover, surface-active adjuvants (e.g., surfactants) are necessary for the production, which are frequently considered as harmful in the case of injection preparations. Further, a control of the concentration behavior in various organs is not controllable in the case of these particles, a linkage of the particles of DE 38 03 972 with selectively accumulating compounds (so-called homing devices, such as, e.g., monoclonal antibodies) is not possible.
The microparticles made of polymerized aldehydes described in DE 40 04 430 are also not suitable as vehicles for substance-specific or structure-specific substances because of the unclear biodegradability. Another drawback is that also in this case surface-active adjuvants are necessary for the production of the particles.
The microparticles made of proteins, especially of albumin, described in EP 0 224 934 exhibit an only very low in vitro and in vivo stability.
It was therefore the object of the invention to provide microparticle preparations especially for use in ultrasonic diagnosis, which get by without the use of physiologically harmful solvents or adjuvants (e.g., surfactants), are easily producible and biodegradable, which either contain substances with site-specific, structure-specific or tissue-specific binding properties in the wall material or can be linked covalently with such and which exhibit a sufficient in vitro and in vivo stability.
According to the invention, this object is achieved by microparticles whose shell is formed from the combination of biopolymersxe2x80x94preferably polypeptides (also glycosylated)xe2x80x94and synthetic material polymerized during the production.
Therefore, microparticles made of a copolymer of at least one synthetic polymer and at least one biopolymer are an object of the invention, and polypeptides, preferably natural ones, or produced synthetically or partially synthetically or obtained by genetic engineering as biopolymers, such as, e.g., albumin, collagen decomposition products, gelatin, fibrinogen, fibronectin, polygeline, oxypolygelatin, their decomposition products as well as poly-L-lysine are suitable. The biopolymers can also be glycosylated. As polymerizable monomers, preferably alkylcyanoacrylates, acrylic acid, acrylamide, acrylic acid chloride and acrylic acid glycide ester are suitable.
The microparticles according to the invention are suitable in the production in gas-saturated solution by the inclusion of the gas, especially as a contrast medium for ultrasonic studies. They act as highly effective scatter elements in the ultrasonic field because of the contained gas. In addition, they can be excited by diagnostic ultrasound to radiate independent signals, which can be evaluated, e.g., with the help of the color Doppler technology.
As gases, air, nitrogen, carbon dioxide, oxygen, helium, neon, argon, krypton or their mixtures are suitable. The charge with the corresponding gas or gas mixture takes place by production of the particles in an aqueous solution saturated with the respective gas or gas mixture.
The microparticles can also (optionally in addition) contain other substances, detectable with the help of medicinally-diagnostic processes, such as magnetic resonance tomography, magnetic resonance spectroscopy, scintigraphy or highly sensitive magnetic field measurements with suitable magnetometers (biomagnetism), both microencapsulated and in the wall material and (optionally with the help of suitable substances, such as, e.g., chelating agents) coupled to the wall material. Thus, it is possible, e.g., in using radioactive isotopes, to use the microparticles according to the invention in scintigraphy. Likewise, its use as contrast medium in magnetic resonance tomography, magnetic resonance spectroscopy or in measurements of the magnetic field is possible by the microencapsulation or incorporation in the wall material of suitable para-, superpara-, ferri- or ferromagnetic substances.
Surprisingly, it has been found that in the production of the particles according to the invention (in maintaining sufficient concentrations of biopolymers), the addition of surface-active substances, such as, e.g., surfactants, is not necessary. This represents a decisive advantage in comparison with the previously known production process for microparticles based on synthetic polymers, since the surfactants usually necessary for reducing the interfacial tension and for preventing the particle aggregation are considered as physiologically harmful and therefore are to be removed again from the preparations before the use in the organism up to compatible residue contents.
As a further advantage of the microparticle preparations according to the invention, the varied particle properties that can be matched to the respective use can be mentioned, which are easily controllable by variation of various production parameters. Thus, the pharmacokinetic parameters of the microparticle preparations, (organ distribution, retention period in the circulatory pathway) can be influenced by the selection of the respectively used biopolymers or by changes of the functional groups of the biopolymer (e.g., by acylation with dicarboxylic anhydrides, such as succinic acid, diglycolic acid, glutaric acid, maleic acid or fumaric acid anhydride or by acetylation with monocarboxylic anhydrides, such as acetic anhydride or propionic acid anhydride).
Further, the content of the biopolymer in the wall material can be varied in a wide scope, by which it is possible to influence the period of the biodegradation of the capsule material in vivo and to match it to the desired use. This content can be controlled directly by the portion of the biopolymer in the production solution. Thus, for example, the wall material consists of microparticles according to the invention, made of 55% (M/M) biopolymers, produced according to example 1 from an autoclaved aqueous solution containing 1% (V/V) butylcyanoacrylic acid and 5% gelatin, while with the same use of butylcyanoacrylate with microparticles produced in 2.5% aqueous autoclaved gelatin solution, the wall material consists of 35% (M/M) biopolymers, with microparticles produced in 7.5% aqueous autoclaved gelatin solution, the wall material consists of 65% (M/M) biopolymers.
Surprisingly, the microparticles according to the invention can be freeze-dried without adding other adjuvants such as lactose, mannitol or sorbitol, as they are usually used as skeleton formers for freeze-drying. These skeleton formers are responsible, after drying, for the mechanical destruction of a considerable part of the microcapsules, which then is no longer usable for the imaging. In contrast to this, in the case of the microparticles according to the invention, the biopolymer of the wall material used in excess is used as a skeleton former, by which surprisingly the ratio of intact to destroyed microcapsules is drastically improved. Because of this more favorable ratio, the dose necessary for imaging can clearly be reduced.
But the microparticles according to the invention can alsoxe2x80x94optionally in additionxe2x80x94contain incorporated pharmaceutical active ingredients, by, e.g., the opacifying agent (in the case of contrast media for ultrasonic studies, a gas or gas mixture is involved here) and one or more active ingredients in the particles being microencapsulated. Preferably, the active ingredients can also be incorporated in the wall material with the methods described for the site-specific, structure-specific or tissue-specific substances. If the active ingredients are biopolymers, they can also partially form the wall material themselves, by being used in the production either exclusively or in a mixture with other suitable biopolymers (e.g., gelatin, albumin, fibronectin, poly-L-lysine) as initial material for microparticle preparation with the addition of a polymerizable monomer or oligomer. The special advantage of coupling active ingredients to the biopolymer portion of the capsule material lies in the fact that active ingredients, which, e.g., are bound by peptide bonds to the biopolymer portion of the capsule material, can be released by enzymatic decomposition in vivo.
The microparticles according to the invention are used especially to detect or to treat thromboses and atherosclerotic changes. In this case, the use of antibodies or antibody fragments against fibrin, fibrin-bonding plasma proteins or their partial structures, tissue plasminogen activators or partial structures of them (e.g., type I-homology and doughnut sequences), protein components of lipoproteins (also partial structures) as homing devices can be considered as especially advantageous.
Other fields of use for the microparticles according to the invention can be, e.g., also the diagnosis or the treatment of hormonal functions (in this case, the use of peptide hormones or their modified products with the capability for receptor bonding as homing devices is to be considered as especially advantageous), or the diagnosis or treatment of lesions of vascular endothelia (in this case, either the use of antibodies or antibody fragments against substances of the integrin group, especially the selectins such as, e.g., LAM-1, ELAM-1 and GMP140, or the use of receptors or their bond-imparting fragments for substances of the integrin group, especially the selectins such as, e.g., LAM-1, ELAM-1 and GMP-140, as homing devices is to be considered as especially advantageous). Moreover, the microparticles according to the invention can also be used for diagnosis or treatment of tumors, by antibodies or antibody mixtures being used as homing devices against surface antigens of tumors.
The production of microparticles according to the invention takes place by the polymerization of a suitable reactive monomer or oligomer (e.g., cyanoacrylic acid butyl ester, cyanoacrylic acid isobutyl ester, cyanoacrylic acid isopropyl ester, cyanoacrylic acid propyl ester, cyanoacrylic acid isohexyl ester, cyanoacrylic acid hexyl ester, cyanoacrylic acid methyl ester, acrylic acid, acrylamide, acrylic acid glycide ester, acrylic acid chloride) in a concentration relative to the total volume of the production solution of 0.01-10% (m/V) (preferably 0.1-10%) under suitable conditions (e.g., by selection of the pH, by adding radicals, and by UV irradiation) with dispersion in aqueous phase, which contains a biopolymer, e.g., albumin, gelatin, oxypolygelatin, polygeline, fibronectin, poly-L-lysine dissolved in a concentration of 0.5-20% (m/V) (preferably 1%-15% (m/V)). By using collagen decomposition products, such as, e.g., gelatin, polygeline or oxypolygelatin, it is often advantageous to autoclave the solutions before the microparticle production. After completion of the polymerization, the resulting microparticles are separated depending on density and particle size by one-time or repeated centrifuging, filtration or flotation, optionally further purified by dialysis and suspended in a physiologically compatible suspending agent (preferably water for injection purposes) until the desired concentration. The suspensions can be isotonized by the addition of suitable water-soluble substances, such as, e.g., glucose, mannitol, sorbitol, common salt, galactose, lactose, fructose, trehalose.
The size distribution of the microparticles developing in the production can be controlled within wide ranges by the type of stirring device used and the number of revolutions.
The production of gas-filled microparticles takes place by the reaction being performed in a solution saturated with the desired gas. In this connection, the density of the resulting microparticles, i.e. the ratio between wall material and gas portion, can be controlled both by the stirring conditions and especially by the portion of biopolymers during the production process.
If microparticles are to be obtained, in which the core consists of the same material as the shell, attention must be paid in the production that by the selection of a suitable stirring device and a suitable stirring speed, a foaming of the reaction solution is avoided.
The required ability for combination with site-specific, structure-specific or tissue-specific substances, which are to assure an additional concentration of the microparticles in target fields outside the organs of the RES (homing devices), takes place either by the coupling of the substances to the polypeptides co-forming the shell material, performed before the microparticle preparation or afterwards, with known methods of biochemistry for coupling amino acids (e.g., W. Kxc3x6nig, R. Geiger: Eine neue Methode zur Synthese von Peptiden: Aktivierung der Carboxylgruppe mit Dicyclohexylcarbodiimid unter Zusatz von 1-Hydroxy-benzotriazolen [A New Method to Synthesize Peptides: Activation of the Carboxyl Group with Dicyclohexylcarbodiimide while Adding 1-Hydroxy-benzotriazoles], Chem. Ber. 103, 788-798 (1970)), or in that the microparticles are produced in an aqueous solution. of the site-specific, structure-specific or tissue-specific substance, if the latter represents a polypeptide, so that the substance is used directly as a component of the shell material.
As site-specific, structure-specific or tissue-specific substances that can be coupled to the microparticles or co-forming the shell material, preferably antibodies, conjugated antibodies, hormones (especially peptide hormones), transferrin, fibronectin, heparin, transcobalamin, epidermal growth factors, lipoproteins, plasma proteins as well as their specificity-imparting partial structures and oligopeptides such as RGD, RGDS, RGDV and RGDT are suitable.
As chelating ligands that can be coupled to the microparticles, diethylenetriaminepentaacetic acid or its derivatives are suitable. The linkage of these ligands with the particles takes place in a way known in the art [Hanatowich et al., Science 220 (1983) 613]. Then, the particles are reacted with the desired metal ions to the respective particle-fixed metal complex.
The selection of the used metal ion depends on the desired area of use. In the field of NMR diagnosis, paramagnetic metal ions, preferred according to the invention, of the elements of atomic numbers 21-29 and 57-70, especially gadolinium(III) ions, are used. For the use in scintigraphy, suitable emitters of radioactive radiation, preferably 111In or 99mTc, 123I and 131I are used.
The finished microparticle suspensions can be used directly for the respectively predetermined use, but it has proven advantageous to improve the storage stability, to freeze and then to freeze-dry the suspensions while adding skeleton formers (such as, e.g., trehalose, polyvinylpyrrolidone, lactose, mannitol, sorbitol, glycine), which also can be used to set the tonicity. It has proven especially advantageous to use the biopolymer used in excess itself as skeleton former. In both cases, it is suitable to move the suspensions during the freezing to prevent uneven particle distributions in the frozen material by sedimentation or flotation. The production of ready-to-use, injectable suspensions from the freeze-dried preparations takes place by resuspending the lyophilizate in a pharmaceutically acceptable suspension medium such as, e.g., water p.i., aqueous solutions of one or more inorganic salts such as physiological electrolyte solutions, aqueous solutions of monosaccharides or disaccharides such as glucose or lactose, sugar alcohols such as mannitol, but preferably in water suitable for injection purposes. The total concentration of the optionally dissolved substances is 0-20% by weight.